First Report of <i>Nectriella adonidis</i> Causing Root Rot of <i>Lespedeza chinensis</i> in China

In August 2023, a root rot disease characterized by brown lesions on the roots, with a 100% incidence rate, was observed in Lespedeza chinensis across 3 survey quadrats (0.5 m × 0.5 m each) in natural grasslands of Huan County (37°7′N, 116°49′E), Gansu Province, China. The symptomatic roots were surface sterilized by immersion in 75% ethanol followed by 1% sodium hypochlorite and were then rinsed three times with sterile distilled water, dried, and subsequently placed on potato dextrose agar (PDA) media and incubated at 25°C. A fungus was consistently isolated from diseased roots, and three strains (LYZ1205, LYZ1206, and LYZ1207) were obtained. Koch's postulates were fulfilled for strain LYZ01205 on L. chinensis seedlings. Seeds were scarified by gently rubbing with sandpaper to break physical dormancy, followed by surface-sterilization 30 seconds in 75% ethanol, followed by 1 minute in 2% sodium hypochlorite, then rinsed thrice in sterile distilled water, and germinated on filter paper moistened with sterile water in Petri dishes. Seedlings with roots approximately 5 mm in length were inoculated by gently pressing the root tip against the edge of a single 2-cm-diameter colony actively growing on PDA. Control seedlings were placed on pure PDA medium for germination. Five replicate pots were established for both inoculated and control treatments. After two days, ten seedlings from either the inoculated or control groups were transplanted into each pot containing sterile loam soil. Plants were maintained in a greenhouse environment at 20 ± 2°C with 80% relative humidity. Inoculated plants exhibited cotyledon desiccation and failure of true leaf emergence at 9 days post-transplantation (dpt), with plant mortality reaching 97.5% by 30 dpt. Discoloration and necrotic lesions were distinctly observed on both the crown and roots of washed plants. Control plants remained asymptomatic throughout the experiment. The fungus was successfully re-isolated from symptomatic roots of inoculated plants and confirmed as the inoculated strain based on morphological and molecular analysis, fulfilling Koch's postulates and confirming its pathogenicity to L. chinensis. On PDA medium, the colony is white, with a felty texture covered by abundant short aerial hyphae, with lobate margins, while the reverse is pale luteous with concentric zonation. No conidial structures or sexual morphs were observed on PDA or five tested sporulation media (potato-carrot agar, potato sucrose agar, wheat straw decoction agar, oatmeal agar, corn meal agar). Partial amplification and sequencing for two loci, including internal transcribed spacer rDNA and the large subunit of the nrDNA, were performed using primer pairs ITS1/ITS4 (White et al. 1990) and LROR/LR5 (Vilgalys & Hester 1990; Rehner & Samuels 1994). The three strains (ITS: PV875827, PV875828, and PV875829; LSU: PV875831, PV875832, and PV875833) were definitively identified as Nectriella adonidis through BLAST searches and Neighbor-Joining phylogenetic analysis (ITS-LSU; bootstrap = 100), showing a complete match with reference strains of N. adonidis (CPC 41516). N. adonidis was initially isolated from Adonis vernalis, a species found in the dry meadows and steppes of Eurasia (Crous et al. 2020), and this remains the sole documented occurrence of N. adonidis. This is the first report of N. adonidis infecting L. chinensis in China. This finding establishes a foundational basis for managing root rot in L. chinensis.